The antigen-specific cytotoxicity mediated by cytolytic T lymphocytes (CTL) is an important part of the immune responses. It is the ultimate goal of our studies to reveal the cellular and molecular requirements for CTL effector functions and differentiation. Our studies led us to propose a novel mechanism of regulation of T lymphocyte functions. According to our model, the phosphorylation of extracellular domains of cell surface proteins may regulate cognate lymphocyte cell-cell interactions by affecting the ligand specificity of cell adhesion proteins and antigen recognition molecules in a manner now accepted as a mechanism for the regulation of enzyme-substrate interactions. No definitive demonstration of ectodomain phosphorylation was previously available in any cellular system and our studies provided the first such example by finding the phosphorylation of the T cell antigen receptor (alphabetaTCR) extracellular domains. We used normal T-cells and T cell transfectants that express alphabetaTCR molecules that lack intracellular and transmembrane protein domains. These cells were studied after 32Pi metabolic labeling. It was found that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alphabetaTCR chains constant regions. These results are consistent with a model in which T cell surface proteins that are phosphorylated intracellularly on their ectodomains could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may affect T cells cognate interactions by affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocyte responses.